Different WDR36 mutation pattern in Chinese patients with primary open-angle glaucoma.

Purpose To determine the distribution of WD repeat domain 36 (WDR36) sequence variants in Chinese patients with primary open-angle glaucoma (POAG). Methods One hundred and thirty-five unrelated POAG patients (82 high tension glaucoma [HTG], 42 normal tension glaucoma [NTG], and 11 juvenile-onset POAG [JOAG] patients) and 77 unrelated controls were recruited. All 23 coding exons and splicing junctions of WDR36 were sequenced using BigDye® Terminator v3.1 cycle sequencing kit. Single nucleotide polymorphism (SNP) and haplotype associations were analyzed using PLINK (version 1.04). Results Nineteen sequence alterations were identified, and eight of them were novel including two novel nonsynonymous SNPs (L240V and I713V). Except the common I264V polymorphism, no other previously reported disease-causing or disease-susceptibility mutations were found. The novel I713V mutation was observed in three (3.7%) patients with HTG. One intronic SNP, IVS5+30C>T (rs10038177), showed significantly higher frequency of minor allele T in HTG patients (16.5%) than in controls (1.3%; Odds ratio [OR]=15.0, p=7.9×10−7, Bonferroni corrected p=1.5×10−5). Haplotype GTA, which is composed of rs13153937, rs10038177, and rs11241095, was significantly associated with HTG (OR=22.5, p=0.002, Bonferroni corrected p=0.013). Neither the individual SNPs nor haplotypes of WDR36 were associated with NTG or JOAG (Bonferroni corrected p>0.05). Conclusions Findings in this study suggest WDR36 to be associated with sporadic HTG but not with NTG or JOAG. Our results also suggest a different mutation pattern of WDR36 in the Chinese population from other ethnic populations.

Although WDR36 has been evaluated for association with POAG in several studies [15][16][17][18], the contribution of WDR36 to the occurrence of POAG is still controversial due to inconsistency in reported associations. Further evaluation of this gene in more populations is needed. The purpose of this study is to determine the distribution of WDR36 sequence variants in a cohort of Chinese patients with POAG.
trauma, uveitis, steroid-induced glaucoma, or exfoliation glaucoma), Shaffer grade III or IV open iridocorneal angle on gonioscopy, and characteristic optic disc damage or typical visual field loss by Humphrey automated perimeter with the Glaucoma Hemifield test. Intraocular pressure (IOP) was determined by applanation tonometry. Control subjects were recruited from people who attended the clinic for conditions of senile cataract, floaters, refractive errors, or itchy eyes. They were excluded from glaucoma using the same criteria of diagnosis as the POAG patients after going through the same procedure of ophthalmic examination. The project was approved by the Ethics Committee for Human Research at the Chinese University of Hong Kong. Informed consent was obtained from all study subjects after explaining the nature and possible consequences of the study in accordance with the tenets of the Declaration of Helsinki.
A cohort of 135 unrelated patients with POAG and 77 unrelated control subjects without glaucoma were included in this study. The demographic and clinical features of the study subjects are summarized in Table 1. The POAG group comprised 110 males and 25 females. Their age at diagnosis ranged from 16 to 85 years (mean±SD: 61±15.0 years). The highest IOP ranged from 13 to 77 mmHg (mean±SD: 24±8.0 mmHg). The vertical cup-disc ratio from 0.7 to 1.0 (mean±SD: 0.8±0.09) and visual field loss were compatible with glaucoma in two consecutive Humphrey testing. In this POAG group, 11 patients were juvenile-onset POAG (JOAG) whose age at diagnosis was less than 35 years (mean±SD: 26.5±6.3 years), the highest IOP ranged from 24 to 77 mmHg (mean±SD: 32.5±7.8 mmHg), 82 patients had late-onset HTG with the highest IOP being greater than or equal to 22 mmHg (mean±SD: 25.7±5.6 mmHg), and 42 patients had late-onset NTG with the highest IOP being less than 22 mmHg (mean ±SD: 17.9±2.3 mmHg). The control group had 58 males and 19 females whose age at inclusion ranged from 52 to 86 years (mean±SD: 72±8.5 years), and their highest IOP ranged from 8 to 21 mmHg (mean±SD: 16±3.0 mmHg), their vertical cupdisc ratio from 0.2 to 0.5 (mean±SD: 0.4±0.07), their visual fields within normal range, and they had no family history of glaucoma.
All the subjects were Han Chinese living in Taiwan. They were recruited from the same eye clinic and had a similar ethnic background. The cases and controls were matched for sex with 81.5% and 75.3% being males in POAG patients and controls, respectively (p=0.29). Because of the age dependence of POAG, only controls older than 50 years of age were included. Mutation screening: Genomic DNA was extracted from 200 µl of whole blood using a commercial kit (Qiamp Blood Kit; Qiagen, Hilden, Germany). Quantification of extracted DNA was performed using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). All 23 coding exons and splicing sites of WDR36 were amplified by polymerase chain reaction (PCR) followed by DNA sequencing. Previously reported primers [22] were used to obtain the initial amplicons. Initial PCRs were performed Cycling conditions were as follows: first denaturation step of 12 min at 94 °C, 35 cycles of denaturation (94 °C for 40 s), annealing (primer-specific annealing temperature for 60 s), elongation (72 °C for 40 s), and a final single elongation step of 7 min. The PCR products were electrophoresed on 2% agarose gel and visualized using a video gel documentation system (Gel-Doc 2000; Bio-Rad Laboratories, Hercules, CA) to check for the quality. The PCR products were then purified with ExoI-SAP kit (USB Corp., Cleveland, OH) to remove unconsumed dNTPs and primers. A second PCR was performed using the sequencing primers as previously described [22] on a thermal cycler (model 9700; ABI) to incorporate the sequencing dyes (BigDye ® Terminator v3.1 cycle sequencing kit; ABI) using a protocol of 25 cycles of denaturation (96 °C for 10 s), annealing (50 °C for 5 s), and elongation (60 °C for 4 min). Sequence data were then aligned using Sequence Navigator analysis software (version 1.0.1; ABI) and compared with the published WDR36 sequence (NM_139281). MYOC and OPTN were screened for sequence alterations by PCR and direct sequencing as previously described [14,23]. Statistical analysis: Statistical analyses were performed using PLINK (version 1.04). PLINK is a free statistical analysis toolset, designed to perform a range of basic and large-scale analyses for genome-wide association studies in a computationally efficient manner [24]. Hardy-Weinberg equilibrium was assessed using an exact test [25]. The frequencies of the WDR36 variants between patients with HTG, NTG, or JOAG and controls were compared using Fisher's exact test. Linkage disequilibrium (LD) analysis was performed using Haploview (version 4.1) [26]. Haplotype frequencies were estimated using the standard E-M algorithm and tested using χ 2 test. Omnibus p values were obtained from the omnibus tests. Specific p values were obtained from the haplotype-specific tests. The odds ratio (OR) and 95% confidence interval (CI) were calculated for each individual haplotype compared to all the other haplotypes. Multiple comparisons were corrected using the Bonferroni method. Despite the Bonferroni correction being considered conservative, especially for small samples, we used it to reduce the possibility of false-positive results and to report our significant association of WDR36 with HTG with more confidence. Disease-causing mutations were defined (1) to alter the amino acid sequence of the corresponding protein and (2) to be completely absent from the control population or significantly more common in the POAG population [27].

RESULTS
Nineteen single nucleotide polymorphisms (SNPs) were identified, eight of which were novel ( Table 2). All SNPs followed Hardy-Weinberg equilibrium in the control group (p>0.05). Three were nonsynonymous SNPs. Two were novel (L240V and I713V) while I264V had been previously reported [15]. The only likely disease-causing mutation, I713V, was found in three patients with HTG (3.7%) and was absent in patients with NTG or JOAG and controls (p=0.25). The L240V variant was found in one control subject (p=0.48). The previously reported common SNP, I264V (rs11241095), was evenly distributed between HTG, NTG, JOAG, and control groups (p>0.51). Three synonymous SNPs, one of which was novel (T180T), and 13 intronic SNPs were found, including five that had not been previously reported ( Table 2). None of the intronic changes or synonymous SNPs was expected to affect splice sites. The minor allele T of IVS5+30C>T (rs10038177) was found in a significantly higher frequency in HTG patients than in controls (p=7.9×10 −7 , Bonferroni corrected p=1.5×10 −5 ; OR=15.0, 95%CI: 3.50, 64.2). The allele T carriers (genotypes TT/CT) had an increased HTG risk (p=2.3×10 −5 ; OR=12.9, 95% CI: 2.91, 57.2) compared to non-allele T carriers (genotype CC). None of the other SNPs of WDR36 was found to be associated with HTG, NTG, or JOAG (p>0.05).

DISCUSSION
In the present study, three patients with HTG (3.7%) carried a novel disease-causing mutation (I713V). They were free of disease-causing mutations in MYOC and OPTN [28]. The absence of this mutation in 154 human normal control chromosomes suggests that it might affect the normal function of the WDR36 protein. This mutation is located within a domain of WDR36 named mini-chromosome maintenance (MCM) protein 5 (amino acids 703-718 and 873-885). MCM proteins are a family of eukaryotic replication factors required for the initiation of DNA replication. MCM5 directly interacts with the Stat1 protein (signal transducer and activator of transcription) to enhance Stat1-mediated transcription activation [29]. These findings suggest that WDR36 might be involved in transcription activation. However, the replacement of an isoleucine by a valine would only mildly alter the hydrophobicity in this region. The conformational structure of the protein is unlikely to be disrupted. It is also possible that the I713V mutation is a benign polymorphism that does not affect the function of WDR36. Further functional studies are required to elucidate the exact role of this novel mutation.
The total mutation prevalence of WDR36 in Chinese patients with HTG (3.7%) is similar to those in the original study of a Caucasian population from USA (3.2%) [15] and one report from Germany (3.7%) [18]. However, it is lower than that in another study of the USA Caucasian population (17%) [16], and higher than the prevalence found in one report of the Japanese population (0.7%) [17]. It is noteworthy that   One disease-causing mutation, I713V, was found in three patients with HTG (3.7%) and was absent in patients with NTG or JOAG and in controls. The minor allele T of IVS5+30C>T (rs10038177 ) was found in a significantly higher frequency in HTG patients than in controls (p=7.9×10 −7 neither individual SNPs nor haplotypes in WDR36 were associated with NTG in our study while WDR36 mutations had been identified in both HTG [15,16,18] and NTG patients [15,18,19] in Caucasian populations. Intriguingly, the similar results of negative association of WDR36 with NTG were also reported in a Japanese population [17], suggesting that the variants of WDR36 may affect only HTG in Asian populations. In the present study, the association analysis was used for the first time to explore ancestral alleles in WDR36 that may pose risk to glaucoma. We found that one intronic SNP of WDR36 (IVS5+30C>T) was significantly associated with HTG (p=7.9×10 −7 ). But we did not find significant association of this variant with the highest recorded IOP and vertical cupdisc ratio (p>0.2; data not shown). Conflicting association of SNP IVS5+30C>T with glaucoma has been reported. Analysis of the published genotype data from a study of a USA population revealed that this SNP was significantly associated with HTG and JOAG (p=1.8×10 −9 ) [16]. However, another study of a German population did not demonstrate significant association of this SNP with POAG (p=0.27) [18]. Our recent study of a northern Chinese population also did not identify significant association of this SNP with HTG and JOAG (p=0.22) [30]. Unfortunately, we could not directly compare these data with our results as the HTG patients were not separately analyzed in those studies [16,18,30]. Notably, we did not find functional mutations in WDR36 in linkage disequilibrium with this common intronic SNP. The only potential disease-causing mutation (I713V) identified in the present study is a rare mutation, which is clearly not the source of this significant association. It is possible that the true disease-causing mutations might be located in the promoter or introns of WDR36 that were unable to be detected in this study. Another possibility is that SNP IVS5+30C>T might be in linkage disequilibrium with disease-causing mutations in a neighboring gene. This latter proposition has some indirect supportive evidence. Two studies reported WDR36 not associated with POAG in several families, which were linked to GLC1G [15,31]. Also, no association was found between WDR36 and unrelated patients with POAG in some populations [20,21]. Although the sample size was not large in our study, it did not compromise the significant association of SNP IVS5+30C>T with HTG. However, other POAG associated sequence variants could still be missed. A largescale study is warranted. The function of WDR36 was recently determined using zebrafish models [32]. It is the functional homolog of yeast U3 small nucleolar RNA-associated protein 21 (Utp21), which is cell essential and functions in the nucleolar processing of 18S rRNA [32]. In yeast models, certain Utp21 variants homologous to glaucoma-associated variants in human WDR36 cause functional defects in a stress-inducedphosphoprotein 1 (Sti1) mutant background, arguing that WDR36 contributes to polygenic forms of glaucoma [33]. Loss of Wdr36 function leads to an activation of the p53 stress response pathway while p53 may act as a transcriptional activator [32]. Collectively, WDR36 might be involved in transcription activation either through its MCM5 domain or through the p53 stress response pathway [29,32]. Although genetic studies of p53 variants have shown inconsistent association with POAG [34][35][36][37], it has been suggested that coinheritance of defects in p53 pathway genes may influence the impact of WDR36 variants on POAG [32]. WDR36 may affect the disease severity of patients with POAG that is caused by mutations in MYOC [16]. We found that OPTN may interact with MYOC and contribute to the development of POAG [38]. This finding was further supported by an in vitro study of human trabecular meshwork cells that OPTN overexpression induced an upregulation of MYOC [39]. Although WDR36 has been shown to function in 18S rRNA processing and transcription activation [32], the exact role of WDR36 in glaucoma remains unclear. It has been suggested that WDR36 may participate in T-cell activation [40]. T-cell responses may be involved in optic nerve degeneration in glaucoma [41]. These findings indicate that WDR36 may contribute to glaucoma by modifying optic nerve degeneration. However, further studies are needed to address how WDR36 may influence POAG.
In summary, our data suggests the association of WDR36 with sporadic HTG but not with NTG or JOAG. Our results also suggest a different mutation pattern of WDR36 in the Chinese population from other ethnic populations.